Download Dr-I-PCMD Lecture Apr-12 - International Center for Chemical

ANTIOXIDANTS
in health and diseases
MUHAMMAD IQBAL CHOUDHARY
Dr. Panjwani Center for Molecular Medicine and Drug Research
International Center for Chemical and Biological Sciences
University of Karachi, Karachi-75270
Oxidation and Human Health
One of the paradoxes of life on this planet is
that the molecule that sustain aerobic life,
oxygen, is not only fundamentally essential for
energy metabolism and respiration, but
implicated in many diseases and degenerative
conditions.
Marx, Science, 235, 529-531 (1985).
Learning Objectives
Understanding the relationship between oxidative
stress, and health and diseases…
• What are oxidants or ROS, types, sources, and activities?
• Their role in normal physiological process
• Their detrimental role in the onset and progression of diseases,
chemical basis of oxidative damage!
• Biomarkers of oxidative damage to the vital biomolecules and
their analysis
• What are antioxidants, types, sources and activities?
• Perceived role of antioxidants in the preservation of health and
prevention of diseases
• Anti-oxidant drug development- challenges and opportunities
CONTENT
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What is oxidation?
Oxidation in biological system
What are free radicals?
Sources of free radicals
Harmful effects of free radicals
Damage to proteins and associated diseases
Damage to DNA and associated diseases
Damage to lipids and associated diseases
Damage to carbohydrates and associated diseases
What are antioxidants?
Nature’s antioxidants system
Dietary sources of antioxidants
Oxidative Stress- Imbalance between oxidation and anti-oxidation
Types of antioxidants
Mechanism of anti-oxidation
Bioassays used to discover new antioxidants
Why This Topic?
If you search for Antioxidants…
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Sci-Finder
Pubmed
Google Search
Chemical Abstracts
248,724 Articles (April 2012)
340,354 Hits (April 2012)
30,500,000 Web pages (April 2012)
14,609 Publications (2011 alone)
What is Oxidation?
• Combination of substrate with oxygen.
• Reaction in which the atoms in a compound
lose electrons.
• Any compound, including oxygen, that can
accept electrons is an oxidant or oxidizing
agent (pro-oxidant), while a substance that
donates electrons is a reductant or reducing
agent (antioxidant).
Oxidation in Biological System
• We live in an aerobic environment
• Oxygen is the life sustaining element
• We consume approximately 3.5 kilograms of
oxygen every day
• 2.8 percent of the oxygen is not properly
used and forms free radicals
• Several kilograms of peroxides (harmful
oxidized lipids) are produced in our body
every year
What are Free Radicals?
• Free radicals (pro-oxidants) are any chemical
species, capable of independent (although
extremely short) existence with one or more
unpaired electrons
• Highly unstable and reactive
• Looking for electrons from other sources to
stabilize themselves. In this process they initiate a
chain reaction of oxidation
• Most commons are Reactive Oxygen Species (ROS)
• Reactive Nitrogen Species (RNS) (NO., ONOO-,
etc) or Reactive Sulfur Species (RSS)
What are Reactive Oxygen Species
(ROS)?
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ROS are:
a.
b.
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oxygen derived radicals (O2.-, .OH, ROO., 1O2,
RO.)
oxygen-derived non radical species (O2, H2O2,
O3, ROOH, HOCl)
They are now considered as major players in
biochemical reactions, cellular response, and
clinical outcome
Triplet Oxygen
(ground state)
O O
Singlet oxygen
O O
Superoxide
O O
Perhydroxyl Radical
O O H
Hydrogen Peroxide
H O O H
Hydroxyl Radical
H O
Hydroxyl Ion
H O
Water
H O H
O O:
+22
O O
O O:
O O:
+7.6
-21.7
H : O-O : H
FREE ENERGY
Kcal / mol
-88
H:O +H:O:H
-53.7
H:O:H
Common Free Radicals
Oxidant
Description
•O2-, superoxide
anion
One-electron reduced state of O2, formed in many
autoxidation reactions and by the electron transport
2+
chain. Rather unreactive but can release Fe from
iron-sulfur proteins and ferritin. Undergoes
dismutation to form H2O2 spontaneously or by
enzymatic catalysis and is a precursor for metalcatalyzed •OH formation.
H2O2, hydrogen
peroxide
Two-electron reduction state, formed by
dismutation of •O2- or by direct reduction of O2.
Lipid soluble and thus able to diffuse across
membranes.
•OH, hydroxyl
radical
Three-electron reduction state, formed by Fenton
reaction and decomposition of peroxynitrite.
Extremely reactive, will attack most cellular
components
Common Free Radicals
Oxidant
Description
ROOH, organic
hydroperoxide
Formed by radical reactions with cellular
components such as lipids and nucleobases.
RO•, alkoxy and
ROO•, peroxy
radicals
Oxygen centered organic radicals. Lipid forms
participate in lipid peroxidation reactions. Produced
in the presence of oxygen by radical addition to
double bonds or hydrogen abstraction.
ONOO-,
peroxynitrite
Formed in a rapid reaction between •O2- and NO•.
Lipid soluble and similar in reactivity to
hypochlorous acid. Protonation forms peroxynitrous
acid, which can undergo homolytic cleavage to form
hydroxyl radical and nitrogen dioxide.
Source= Wikipedia
What Free Radical does?
Free radicals are cellular renegades; they wreak havoc
by damaging DNA, altering biochemical compounds,
corroding cell membranes and killing cells outright.
Such molecular mayhem, scientists increasingly
believe, plays a major role in the development of
ailments like cancer, heart or lung diseases and
cataracts. Many researchers are convinced that the
cumulative effects of free radicals also underlie the
gradual deterioration that is the hallmark of aging in
all individuals, healthy as well as sick.
TIME, April 6, 1992 publications
Internal Sources of Free Radicals
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Mitochondria
Phagocytes (macrophages)
Xanthine oxidase
Reaction involving iron and other transition metals
Arachidonate pathways
Peroxisomes
Exercise
Inflammation
Ischaemia/Reperfusion
External Sources of Free
Radicals
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Cigarette smoke
Environmental pollutants
Radiations
Ultraviolet radiations
Ozone
Certain drugs, pesticides, anesthetics and
industrial solvents
Useful Functions of Free
Radicals
• Necessary in the maturation processes of cellular
structures
• Necessary in the antibacterial activity- White blood
cells (phagocytes) releases free radicals to destroy
invading pathogenic microbes as part of the body’s
defense mechanism
• Necessary in the immune system
• Necessary in the prostaglandin biosynthesis
• Some of them play an important role in cell
signaling
History: Harmful Effect of
Free Radicals
• 1775 Priestly- Toxicity of oxygen to the
organism similar to burning of candle
• 1954 Gilbert and Gersham- Free radicals are
important player in biological environment
and responsible for deleterious process in the
cell
• 1969 Mc Cord and Fridovich- Superoxide
theory of toxicity
Harmful Effect of Free
Radicals- Perception
• Free radicals can damage all cellular
macromolecules
including
proteins,
carbohydrates, lipids and nucleic acids
• Destructive effect play a role in the onset and
progression of different diseases and in
normal aging.
Oxidative Damage to
Organs
Ageing is one of the major
consequences of oxidative damage
1956 Denham- Free Radical Theory of Ageing
Human Ailments Associated with
Oxidative Damage
Neurological
Alzheimer’s Disease
Parkinson‘s Disease
Endocrine
Diabetes
Gastrointestinal
Acute Pancreatitis
Human Ailments Associated with
Oxidative Damage
Others Conditions
Obesity
Loss of catalytic functions of proteins
Toxicity
Chronic Inflammation and arthritis
Diseases Related to Oxidative Damage
Exogenous v/s Endogenous
Sources of Free Radicals
• Exogenous ROS are extremely high
• Exposure to endogenous oxidants is much more
important and extensive, because it is a continued
process during the entire life span
• Mitochondria play an extremely important role in
endogenous ROS production
• Presence of metals (iron, copper, chromium,
cobalt, vanadium) in un-complexed form
significantly increase the level of oxidative stress.
Damage to Lipids
• Lipids are highly prone to get oxidized.
• Polyunsaturated fatty acid (PUFA)- major
part of the low-density lipoprotein (LDL) in
blood.
Damage to Lipids
• Malondialdehyde (MDA) is a biomarker of
oxidative stress.
End Products of Lipid Peroxidation
These end products are the
markers for lipid peroxidation
determination. For example
malondialdehyde (MDA) is
detected in TBARS (Thiobarbituric Acid Reactive Substance)
assay, specific for lipid
peroxidation determination.
Damage to Lipids-Associated
Diseases
• Alterations in the structures of lipid molecules lead
to change in their physical properties, such as
permeability, surface adhesion, etc.
• It cause damage to the cell membrane, made up of
mainly lipids.
• Risk of cardiovascular diseases (CVD), including
atherosclerosis.
Damage to Lipids-Associated
Diseases
• End products of lipid peroxidation can also cause
mutagenesis and carcinogenesis. For example
malondialdehyde reacts with deoxyadenosine and
deoxyguanosine in DNA to form a variety of DNA
adducts.
• Body has evolved a range of molecules, such as
Vitamin E, and enzymes such as SOD, catalase,
and peroxidase to control lipid peroxidation.
• Knockout animals (which can not produce antioxidant enzymes), do not survive beyond
embryonic stage.
Atherosclerosis and Oxidative
Stress
• Compelling evidence points oxidative stress as an important
trigger in the complex chain of events leading to
atherosclerosis.
• It involve accumulation of macrophages in the arterial wall.
Which then avidly incorporate oxidized LDL to form foam
cells.
• ROS can lead to platelet activation and thrombosis
formation.
• Probucol has reduced progression of carotid atherosclerosis
in clinical trials.
Oxidative Damage to
Proteins
• .OH and RO. and cause damage to proteins
• Direct damage include peroxidation, damage
to specific amino acid residues, change in
tertiary
structures,
degradation
and
fragmentation
• No efficient mechanism of repair of protein
damage exists
• Proteolytic enzymes play an important role
in the removal of damaged proteins
Protein Oxidation Products
• Aldehydes, keto and other carbonyl compounds
• 3-Nitrotyrosine, produced by interaction of
tyrosine and ONOO-, is a useful biomarker of
oxidative protein damage
• Ortho- and meta-tyrosines from phenylalanine.
• Other damaged products include hydroxyproline,
glutamyl semialdehyde, etc
• Crossed linked proteins
Damage to ProteinsAssociated Problems
• Modified oxidized proteins are susceptible to many
changes in their functions
• This include chemical fragmentation, inactivation
and increased proteolytic degradation
• Oxidative changes in the structures of catalytic
proteins lead to loss of enzyme activity
• Altered cellular functions such as energy
production, interference with the creation of
membrane potential and change in the type and
level of cellular proteins
• Glycation of proteins
Mechanism of Glycation of Protein- Role of
ROS
Glucose + Protein
Schiff
base
Amadori product
Protein Enediol
O2
O2 O2
Mn
OH
Mn
M (n -1)
M (n -1)
H2O2
Protein dicabonyl
Advanced
Glycation
Endproducts
Catalyzed by transition metals (M) and the superoxide radical generated are converted to
the hydroxyl radical via the Fenton reaction.
Oxidative Damage to DNA
• DNA is stable, well protected molecules
• ROS, specially .OH, can interact with it and
cause several types of damage
• Including modification of DNA bases,
single- and double helical breaks, loss of
purines, damage to deoxyribose sugar, DNA
protein cross linkage and DNA repair system
• Out of four bases, guanine is the most easily
oxidizable nucleic acid base.
Oxidative Damage to DNA
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Oxidative Damage to DNA
• Oxidative products of guanosine serve as
biomarkers of damage to DNA molecule.
Oxidative Damage to DNA
• ROS in the cells lead to DNA damage, cause stable
DNA lesions which are mutagenic, if un-repaired
• Damaged DNA provide the wrong genetic code
leading to unregulated protein synthesis and/or
cell growth which results in cancer.
• Presence of 8-oxo-2-deoxyguanosine (oxo8dG) in
DNA is an important indicator of oxidative damage
to DNA
• Oxidative damage to DNA accumulate with ageing,
increasing the possibilities of cancers and other
disorders
Damage to DNAAssociated Problems
• Number of oxidative hits to DNA per cell per day is
about 100,000 in the rat and about 10,000 in the
human (Reason????)
• There is an inherent mechanism (specific repair
glycosylases, etc.) to repair most of the DNA
damage caused by ROS
• Oxidative lesions in DNA accumulate with age and
eventually lead to serious health challenges (well
established relationship between onset of cancers
and age)
Oxidative Stress Markers
Oxidative stress end products detection
Lipoperoxidation markers:
malondialdehyde (MDA), conjugated dienes,
isoprostanes
Oxidative damage to protein markers :
protein hydroperoxides
Oxidative damage to DNA :
modified nucleosides
What are Antioxidants?
• Antioxidants (reductants or reducing agents)
are compounds capable of preventing the
pro-oxidation process or biological oxidative
damage by scavenging or stabilizing reactive
oxidative species.
An antioxidant is a molecule stable enough to donate
an electron to a rampaging free radical and neutralize
it, thus reducing its capacity to damage.
What are Antioxidants?
• Antioxdiants produced during normal metabolism
include glutathione, ubiquinol and uric acid
• Antioxidant
enzymes
include
glutathione
peroxidases, superoxide dismutases and catalase
• Antioxidants from dietary sources such as Vitamins
E and C and carotenoids
• Antioxidants from non-dietary sources include
phenolic or polyphenolic compounds as well as
selenium
What Antioxidant Do???
WHY ARE ANTIOXIDANTS
IMPORTANT ?
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They inhibit the conversion of nitrites to nitrosamines
(which are tumor promoters) and enhance the immune
response.
 Vitamins E, and C, ubiquinones, etc. remove free
radicals from the epidermis of the skin and counteract
their potentially damaging effect.
 They terminate free radical- induced cellular
damage and functional degeneration (aging).
 They trap and neutralize free radicals and protect our
body tissues from environmental pollutants.
Sources of Antioxidants
• More than 4,000 antioxidants are known
• Endogenous- Antioxidant enzymes include
glutatione
peroxidases,
superoxide
dismutases and catalase
• Antioxidants from dietary sources such as
Vitamin E, Vitamin C and carotenoids
• Antioxidants from non-dietary sources
include phenolic or polyphenolic compounds
Antioxidant Enzymes
• Glutathione peroxidases catalyze the
reduction of lipid hydroperoxides to their
corresponding alcohols
• Superoxide dismutases, a family of metalcontaining enzymes (Mn, Fe, Zn, Cu),
catalyze the dismutation of superoxide into
oxygen and hydrogen peroxide
• Catalases catalyze the decomposition of
hydrogen peroxide to water and oxygen
Major AntioxidantsVitamin E
• Vitamin E, fat-soluble vitamin which exists
in eight different forms. a-Tocopherol is the
most active form in humans.
• Vitamin E protect cells through its ability to
limit production of free radicals
• Dietary sources include wheat, almonds,
sunflower seeds, etc.
Major Antioxidants- Vitamin C
• Vitamin C, or L-ascorbate is an essential
nutrient, water-soluble
• Vitamin C scavenge aqueous peroxyl radicals
Major Antioxidants- Carotenoids
• Organic pigments naturally occur in plants
• 600 known carotenoids, and tetraterpenoids exist in
nature
• Most common are lycopene and vitamin A precursor bcarotene
• They quench singlet oxygen primarily by a physical
mechanism in which the excess energy of the singlet
oxygen is transferred to the carotenoids electron rich
structure
Lycopene
Major AntioxidantsPlant Phenolics
• A large number of plant phenolics, such as
flavanoids, and catechins act as free radical
scavengers
• Tea is the richest source of plant phenolics
• French paradox – Flavanoids in red wine
Major AntioxidantsPlant Phenolics
Apigenin
Apigenin
Catechin
Antioxidants at a glance
Nutrient
Vitamin E
RDI
30 IU
Vitamin C
60 mg
Dietary Sources
Vegetable oils (soy, corn, olive,
cotton-seed, safflower, sunflower),
nuts, sunflower seed, wheat germ
Citrus, strawberries, tomatoes,
cantaloupe, broccoli, asparagus,
peppers, spinach, potatoes
ß-Carotene
NA
Selenium
70 ug
55 ug
Evidence
100-800 IU may lower
heart disease risk by
30%-40%
no evidence
that RDI
supplement form can
prevent CHD or
cancer
may protect against
CHD and macular
degeneration
Dark green, yellow, and orange
vegetables: spinach, collard green
broccoli, carrots, peppers, sweet
potatoes; yellow fruits: peaches
Egg yolks, tuna, seafood, chicken, 150-200 ug may lower
liver, whole grains, plant grown in prostate cancer risk
selenium-rich soil.
Balance Between Oxidation and
Antioxidation- Key to Health
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Balance between pro-oxidants and oxidants is tightly
regulated and extremely important for maintaining
vital cellular and biological functions
Oxidative and reductive stress
Oxidant
Reductant
Oxidant
Reductant
Oxidative stress
Reductive stress
What is Oxidative Stress?
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If there are too many free radicals produced and too
few anti-oxidants, a condition of “oxidative stress”
develop which may cause chronic diseases.
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An imbalance between the production of various
reactive species and the ability of the organism’s
natural protective mechanism to cope with these
reactive compounds and prevent adverse effects.
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It has been proven to be related to degenerative
diseases such as cancers, diabetes, premature ageing,
Alzheimer’s disease, arthritis, etc.
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Difficult to measure.
Oxidative Stress Markers
Free radical detection
• Very difficult to analyze because of chemical and
physical properties e.g. short half life
Oxidative stress end products detection (foot
print measurement)
• more simple, a wide range of techniques available
Some Common In Vitro Antioxidant Assays
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NO· ( Nitric Oxide) Radical Inhibition
Assay for DPPH Radical Scavenging Activity
Assay for xanthine oxidase inhibition Activity
Assay for superoxide anion scavenging Activity
a. Enzymatic generation of superoxide anion (through xanthine
oxidase)
b. Non-Enzymatic generation of superoxide anion (through
NADH/Phenazine methosulphate system)
Some Common In Vivo Antioxidant Assays
• In vivo CCl4 (Carbon tetrachloride) hepatoxicity assay.
• Antioxidant testing kits are now available to detects free
radical activity in body.
DILEMA OF REDOX IN
LIVING SYSTEM
• Difficult to quantify the oxidizing stress
• Difficult to extrapolate in vitro and in
vivo situations
• Bioavailability is an issue
• Interaction with other molecules
• Whether antioxidants do help in the
prevention of diseases?
• More questions than answers!!!!
Antioxidant Drug DevelopmentChallenges and Opportunities
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Difficult
THANK YOU VERY MUCH